Partial in vivo reprogramming enables injury-free intestinal regeneration via autonomous Ptgs1 induction

Tissue regeneration after injury involves the dedifferentiation of somatic cells, a natural adaptive reprogramming that leads to the emergence of injury-responsive cells with fetal-like characteristics. However, there is no direct evidence that adaptive reprogramming involves a shared molecular mechanism with direct cellular reprogramming. Here, we induced dedifferentiation of intestinal epithelial cells using OSKM (Oct4, Sox2, Klf4, and c-Myc) in vivo. The OSKM-induced forced dedifferentiation showed similar molecular features of intestinal regeneration, including a transition from homeostatic cell types to injury-responsive–like cell types. These injury-responsive–like cells, sharing gene signatures of revival stem cells and atrophy-induced villus epithelial cells, actively assisted tissue regeneration following damage. In contrast to normal intestinal regeneration involving Ptgs2 induction, the OSKM promotes autonomous production of prostaglandin E2 via epithelial Ptgs1 expression. These results indicate prostaglandin synthesis is a common mechanism for intestinal regeneration but involves a different enzyme when partial reprogramming is applied to the intestinal epithelium.

and quantification of Ki67-positive cells per crypt (-Dox, n = 10; +Dox, n = 13) (right).(E) Violin plots showing log10-scaled value of unique molecular identifiers (UMIs) (left) and log10-scaled value of the number of features in -Dox and +Dox conditions.(F) UMAP plot of scRNA-seq from OSKM-induced mouse intestinal epithelium indicated with 18 distinct clusters.(G) Dot plot for expression of canonical marker genes per each cluster.(H) Bar plot showing cell type proportion in each condition.(I) Box plot indicating signaling entropy rate (SR) inferred by SCENT per each condition in different cell types.(J) Violin plots showing expression of differentiated cell-type marker genes between conditions in different cell types.(K) Violin Plot showing expression of Lgr5 between conditions in CBC.(L) IF of Trop2 in the intestine of iOSKM mice.DAPI for nuclear staining.Data represent the mean with SD.Student's t-test: p < 0.05(*), p <0.01(**), p <0.001(***), ns, not significant.Scale bar = 50µm.

Fig. S3 .
Fig. S3.Characterization of OSKM-induced revSC-like and aVEC-like cells (A) UMAP plots showing conditions (left) and cell types (middle) of revSC-containing scRNA-seq data and projection results of revSC-containing scRNA-seq data onto our scRNA-seq data (right).(B) UMAP plots showing conditions (left) and cell types (middle) of aVEC-containing scRNA-seq data and projection results of aVEC-containing scRNA-seq data onto our scRNA-seq data (right).(C) Relative expression of revSC, aVEC, top villus, EC and fetal gene markers in each cell type, (D-F) Violin plots showing expression for revSC (D), aVEC (E) marker genes, and DC2-specific expressed genes (F).(G) Venn diagrams showing overlapping cell type-specific marker genes between DC1, DC2 and EC.Colors indicating reported revSC-specific (red), revSC/aVEC common (purple) and aVEC-specific (blue) marker genes.(H) Violin plots showing expression for villus-top (left) and villus-bottom (right) EC marker genes.(I) UMAP plots showing gene

Fig. S4 .
Fig. S4.Promoted intestinal regeneration after IR damage by partial reprogramming (A) Western blotting for Oct4 in intestinal epithelial cells of iOSKM mice on Dox treatment for indicated day(s).α-tubulin for loading control.(B, C) Intestine of iOSKM mice after 10Gy IR.Dox was treated 2 days before IR for 4 days.H&E histology at 4dpi (B) and IF of Trop2 at 2dpi and 4dpi (C).(D) Experimental scheme for 5-FU and Dox treatment in iOSKM mice.(E) H&E histology and IHC for Olfm4 and Ki67 in the intestine of iOSKM 4 days after 5-FU treatment.(F) IF of Sca1 and BrdU in the intestine of iOSKM mice 2 days and 4 days after 5-FU treatment (top) and the quantification of BrdU+ cells per crypt (bottom).(n = 2 mice) (G) Caspase-3 activity using intestinal organoids 24 hours after IR (n=2) (H) Microscopic images of control and Dox-treated intestinal organoids until 8 dpi without passaging (left) and the quantification of the number of buds per organoid after IR (right) (I) Microscopic images of intestinal organoids with Dox and IR treatment.The morphology was analyzed on day 9 (2 days after first passage) and day 19 (5 days after second passage) after IR.Data represent the mean with SD.Student's t-test: p < 0.0001(****), ns, not significant.Scale bar = 50µm (C, E, and F), 500µm (H and I).

Fig. S5 .
Fig. S5.Inhibition of fetal gene transition and YAP activation by NSAID in intestinal organoids (A) Bar plots showing enrichR combined scores of top enriched pathways of common upregulated genes in indicated condition.(B) Microscopic images of Dox-treated intestinal organoids with or without NSAID (left) and quantification of budding organoid and spheroid ratio (right) (n =4).(C) IF of Sca1 in mouse intestinal organoids.(D) mRNA expressions of fetal gene Ly6a (encoding Sca1) iOSKM intestinal organoids.(E) IF of active YAP in mouse intestinal

Fig. S6 .
Fig. S6.Increase in Cox1 expression and prime role in intestinal regeneration (A) IF of Cox1

Table S1 .
Gene signatures used in this studyMovie S1.Growth of intestinal organoids in both conditions, control (A) and Dox treatment (B),